Determination of several psychiatric drugs in whole blood using capillary gas-liquid chromatography...

Determination of several psychiatric drugs in whole blood using

capillary gas–liquid chromatography with nitrogen phosphorus

detection: comparison of two solid phase extraction procedures

Carolina Sanchez de la Torre, Marıa A. Martınez*, Elena Almarza

National Institute of Toxicology and Forensic Sciences, Ministry of Justice, C/Luis Cabrera 9, 28002 Madrid, Spain

Received 23 August 2004; received in revised form 4 November 2004; accepted 3 December 2004

Available online 26 January 2005

Abstract

A simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization was developed

for the detection of several psychiatric drugs in whole blood as part of systematic toxicological analyses (STA). Drugs included

mirtazapine, chlorpromazine, methotrimeprazine (levomepromazine), clothiapine, olanzapine, clozapine, haloperidol, and

thioridazine. All drugs were studied at concentrations of 100–2000 mg/L, except haloperidol that was studied at concentrations

of 400–8000 mg/L. In order to select the best blood purification procedure and therefore increase the signal to noise ratio we have

compared two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, for their recovery, precision, sensitivity

and matrix purification efficiency. Recoveries for these drugs using Chem Elut columns at 500 and 2000 mg/L (2000 and

8000 mg/L for haloperidol) were in the range 21–65%, with intra-assay and inter-assay precisions of less than 17% and 19%,

respectively. Limits of detection (LODs) and limits of quantitation (LOQs) for mirtazapine, chlorpromazine, methotrimepra-

zine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 62 to 161 mg/L and from 205 to 531 mg/L, respectively.

LOD and LOQ for haloperidol were 442 and 1458 mg/L, respectively. Recoveries of these compounds using Bond Elut Certify

columns at 500 and 2000 mg/L (2000 and 8000 mg/L for haloperidol) were in the range 44–97%, with intra-assay and inter-assay

precisions of less than 7% and 14%, respectively. LODs and LOQs for mirtazapine, chlorpromazine, methotrimeprazine,

clothiapine, olanzapine, clozapine, and thioridazine ranged from 37 to 66 mg/L and from 122 to 218 mg/L, respectively. LOD

and LOQ for haloperidol were 156 and 515 mg/L, respectively. Linearity was observed in the studied range for all compounds

with r2 values of >0.999. The use of the mixed-mode bonded-silica Bond Elut Certify columns showed advantages comparing

with Chem Elut columns for the screening of these psychotropic agents such as higher recoveries, cleaner extracts, better

sensitivity, better precision and less solvent consumption and subsequent disposal.

# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Systematic toxicological analysis; Psychiatric drugs; Solid phase extraction; GC; Screening

www.elsevier.com/locate/forsciint

Forensic Science International 155 (2005) 193–204

1. Introduction

Psychoses are among the most severe psychiatric dis-

orders, in which there is not only marked impairment of

* Corresponding author.

E-mail address: [email protected] (M.A. Martınez).

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights r

doi:10.1016/j.forsciint.2004.12.007

behavior but also a serious inability to think coherently, to

comprehend reality, or even to gain insight into the presence

of these abnormalities. These common disorders (affecting

perhaps 0.5–1.0% of the population at some age) typically

include symptoms of false beliefs (delusions) and abnormal

sensations (hallucinations). Representative syndromes in

this category include schizophrenia, brief psychoses, and

eserved.

C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204194

delusional disorders, although psychotic features also are

uncommon in the major mood disorders, particularly mania

and severe melancholic depression. Antipsychotic agents are

used primarily in the management of patients with psychotic

or other serious psychiatric illnesses marked by agitation and

impaired reasoning.

Depression is also one of the most common and most

debilitating mental disorders. It is characterized by feelings

of intense sadness and despair, mental slowing and loss of

concentration, pessimism, lack of pleasure, self-deprecation,

and variable agitation. Antidepressant agents, which act

blocking neuronal uptake of biogenic amines, represent

the first choice in the treatment of moderate to severe

depressive illness [1].

Pharmacological treatment is essential for an adequate

management of these psychiatric disorders. Toxicity to

antipsychotics and antidepressants can worsen patient status

and may be associated with elevated blood levels. Drug

interactions can be recognized more readily if routine blood

level monitoring of patients is the rule rather than the

exception. Deliberate and accidental cases of self-poisoning

with these therapeutic drugs have become a major medical

problem because of the widespread use of these therapeutic

drugs and the severity of their toxic actions. This fact has led

to the development of reliable analytical methods for their

analysis.

In systematic toxicological analysis (STA) one of the

main purposes is drug screening. Antipsychotics and anti-

depressants are some of the most frequently encountered

drugs in forensic and clinical cases. In STA the substances

present are not known at the start of the analysis. In such

cases the extraction procedure cannot be directed to a given

substance but must be a general procedure where a com-

promise must be reached so that the substances of interest are

capable of being detected with high recovery, efficiency, and

minimal presence of potential interfering substances. Tra-

ditionally, liquid–liquid extraction (LLE) has been routinely

used in most forensic laboratories. As an extraction method,

solid phase extraction (SPE) is growing in use as a very

effective procedure for sample pre-treatment and clean-up of

biological samples in all types of drug testing laboratories,

including those which carry out forensic toxicology. SPE

offers several advantages over traditional LLE, including

better specificity, the ability to obtain cleaner extracts, good

reproducibility, avoidance of emulsion formation, the ability

to automate the extraction procedure, and substantial reduc-

tion in the volume of solvent required [2].

There are numerous publications on SPE for relatively

clean biological samples such as plasma, serum or urine

[3–12]. Until now, only a few publications have described

SPE methods for whole blood [13–23]. However, hemolyzed

whole blood is in practice the matrix most frequently

encountered in forensic toxicology. It is worth mentioning

that, post-mortem whole blood is not as fluid as ante-mortem

serum or plasma and therefore generally requires more

workup.

The Chem Elut column, a diatomaceous earth support, is a

SPE procedure closely related to conventional LLE. The

aqueous sample is added to the top of the column, adsorbed,

and distributed into a film over the diatomaceous earth, which

is an hydrophilic packing porous material which acts as

support for the aqueous phase. The high surface area of the

inert diatomaceous earth ensures that the organic eluents

remain immiscible with the aqueous matrix, eliminating

emulsion problems, and facilitating efficient interaction

between the sample and the organic solvent [24,25]. However,

for STA purposes where acidic, neutral, and basic substances

may be present, this type of SPE must be carried out with at

least two columns: one for the acidic and neutral substances

and one for basic and neutral substances and often large

volumes of hazardous organic solvents are required [2].

The Bond Elut Certify column, a mixed-mode bonded

silica SPE extraction support containing hydrophobic chains

(octylsilane, n-C8) and strong cation-exchange moieties (ben-

zenesulphonylpropylsilane), has the ability to retain acidic,

neutral, basic and amphoteric drugs on one column. This is a

great advantage in the field of toxicology where blood speci-

mens available for analysis are sometimes very small and a

search of a very wide variety of drugs is required [20,22,23].

The use of capillary gas–liquid chromatography using

fused-silica capillary columns and nitrogen-phosphorus

detection (GC-NPD) has proved to be a powerful tool in

the area of underivatized drug analysis in biological extracts.

Since many drugs are nitrogen-containing compounds,

traces of them can be selectively detected in the screening

toxicological analysis with excellent sensitivity and minimal

interference from other non-nitrogenous compounds, both

endogenous and exogenous [26]. This is crucial for whole

blood drug analysis considering that it is not a clean matrix

and the concentrations of the drugs or metabolites are much

smaller compared with urine.

This paper presents a rapid and sensitive validated

method which allows the simultaneous determination of

whole blood therapeutic and/or toxic concentrations of

the antidepressant mirtazapine and the antipsychotics: chlor-

promazine, methotrimeprazine (levomepromazine), clothia-

pine, olanzapine, clozapine, haloperidol, and thioridazine,

using GC-NPD analysis without derivatization (see Fig. 1).

Mepivacaine and prazepam were used to control the whole

analytical procedure. Two SPE analytical procedures were

compared, Chem Elut and Bond Elut Certify columns, for

their recovery, precision, sensitivity, and matrix purification

efficiency.

2. Experimental

2.1. Materials

Mirtazapine was donated by Organon (Roseland, NJ,

USA), clothiapine was donated by Novartis Pharma AG

(Basel, Switzerland), and olanzapine was donated by Eli

C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204 195

Fig. 1. Chemical structures of the eight psychotropic studied in this work and the compounds used to control the analytical procedure.

Lilly (Dunderrow, Kinsale Co., Cork, Ireland). Chlorproma-

zine hydrochloride, methotrimeprazine maleate, clozapine,

haloperidol, thioridazine hydrochloride, mepivacaine hydro-

chloride, and prazepam were purchased by Sigma–Aldrich

(Buch, Switzerland).

Individual stock solutions were prepared by dissolving

10 mg of each pure compound in 10 mL of methanol. These

stock solutions were stored in glass tubes and maintained at

�27 8C. Appropriate dilution with methanol yielded the

working solutions containing all compounds involved in

the study. The extraction standard solution was prepared

by diluting the stock solution of mepivacaine with deionized

water to 16 mg/L. The chromatographic standard solution

was prepared by diluting the stock solution of prazepam with

methanol to 5 mg/L.

All chemicals (Merck, Darmstadt, Germany) and sol-

vents (Scharlau, Barcelona, Spain) were of analytical grade.

Borate buffer (saturated solution, pH 9.0) and dichloro-

methane–isopropanol (85:15) were used for the Chem Elut

procedure. Phosphate buffer (0.1 M, pH 6.0), 0.01 M acetic

acid, acetone–dichloromethane (1:1), and dichloromethane–

isopropanol–ammonia (78:14:8) were used for the Bond Elut

Certify procedure.

Chem Elut CE 1010 columns (10 mL of column reservoir

volume), Bond Elut Certify columns (130 mg of sorbent

mass, 3 mL of column reservoir volume) and a VAC-ELUT

SPS 24 vacuum manifold system for the manual mixed-

mode bonded silica SPE were purchased from Varian Sam-

ple Preparation Products (Harbor City, CA).

A P-Selecta sonication bath and a P-Selecta Centronic S

centrifuge were both purchased from Selecta (Barcelona,

Spain).

A pool of citrated human whole blood samples was

provided by Comunidad de Madrid Blood Bank (Madrid,

Spain) and verified to be drug free. No interferences were

found for the studied compounds and the samples were kept

frozen at �27 8C until used.

2.2. Instrumentation

The analysis of the extracts was performed on a Hewlett-

Packard (HP) (Avondale, PA) model 5890 Series II gas

chromatograph equipped with a nitrogen-phosphorus detec-

tor and linked to a HP 3396A integrator. The column was a

25 m � 0.20 mm i.d. fused silica capillary column coated

with cross-linked methylsilicone (0.11 mm film thickness)


(HP Co., PA). The following temperature program was used

with a total run time of 18 min, including 2 min of equili-

bration time. The initial column temperature was set to

180 8C for 1 min, and then increased to 300 8C at 10 8C/

min. This final temperature was held for 3 min. The carrier

gas was helium (Air Liquid, Madrid, Spain) delivered at a

column head pressure of 195 kPa. Injection port and detector

temperatures were 280 and 300 8C, respectively. The NPD

was operated at a baseline of 30–35 pA. The splitting ratio

was 1:20.

Deactivation of insert liners were performed according to

the following procedure traditionally used in our laboratory.

Insert liners were silanized with dimethyldichlorosilane:to-

luene (5:100) after standing in contact with the solution for

30 min. Excess dimethyldichlorosilane was rinsed away

with toluene:methanol (90:100) after soaking for 30 min

and the process was completed with an acetone rinse of

30 min. After removing the excess of solvent, they were

packed with Supelco silanized glass wool (from Supelco

Park, Bellefonte, PA). In order to minimize a reduction in the

performance of the chromatographic system, it is essential to

ensure that insert liners are kept clean. Routinely, they were

replaced before each batch of injections (approximately 100

injections).

2.3. Preparation of spiked whole blood samples

Human whole blood (100 mL) was spiked with mirta-

zapine, chlorpromazine, methotrimeprazine, clothiapine,

olanzapine, clozapine, and thioridazine at three concentra-

tion levels 0.1, 0.5 and 2 mg/L, respectively, while the

respective concentrations for haloperidol were 0.4, 2 and

8 mg/L. The spiked blood was sonicated for 15 min at room

temperature and then it was submitted in parallel to SPE

using Chem Elut and Bond Elut Certify columns.

2.4. Chem Elut extraction procedure

The extractions were performed using a Chem Elut

extraction procedure described in our previous work [20].

Briefly, each aliquot (2 mL), was treated with 7.0 mL of

borate buffer (pH 9.0) and 100 mL of a mepivacaine

aqueous solution of 16 mg/L as extraction standard, vortex

mixed for 5 min, and loaded onto the Chem Elut columns.

After 5 min, elution of the sample by gravity was carried

out adding two aliquots of 10 mL of dichloromethane–

isopropanol (85:15). Eluates were evaporated under a

nitrogen stream. The extraction residues of blood were

reconstituted with 200 mL of methanolic solution of chro-

matographic standard (5 mg/L) and 2 mL were injected for

GC analysis.

2.5. Bond Elut Certify extraction procedure

The extraction procedure with Bond Elut Certify col-

umns was also described in our previous work [20]. It was

performed on a VAC-ELUT SPS 24 vacuum manifold

system. To each 2.5 mL aliquot, 7.5 mL of phosphate buffer

(pH 6.0) and 125 mL of a mepivacaine aqueous solution of

16 mg/L as extraction standard were added, sonicated for

5 min and centrifuged at 4000 rpm for 10 min, then 8 mL of

the supernatant, equivalent to 2 mL of whole blood, were

used for further extraction. The columns were precondi-

tioned with 1 mL methanol, followed by 1 mL 0.1 M phos-

phate buffer (pH 6.0) under light vacuum (approximately

2 in. Hg) to avoid the columns becoming dry before the

application of the sample. The whole blood was applied onto

the columns and drawn through completely at a flow rate of

approximately 1.5 mL/min. The columns were washed with

2 mL deionized water. The columns were acidified by

passing through 0.5 mL of 0.01 M acetic acid. Then the

columns were dried under full vacuum (15 in. Hg) for 4 min.

Methanol (60 mL) was added, and the columns were dried

under full vacuum for 1 min.

After the column outlets were wiped with tissue, the

labelled evaporation tubes recently rinsed with methanol,

in order to avoid retention of polar drugs in the walls of the

glass tubes, were placed into the manifold basin. To each

column, first 3.5 mL of acetone–dichloromethane (1:1)

was added, and after 3 mL of dichloromethane–isopropa-

nol–ammonia (78:14:8). The eluents were pulled through

completely at a flow rate of 0.8 and 0.5 mL/min, respec-

tively and the combined eluates were evaporated under a

nitrogen stream. The extraction residues of blood were

reconstituted with 200 mL of methanolic solution of chro-

matographic standard (5 mg/L) and 2 mL were injected for

GC analysis.

2.6. Validation of the methods

The spiked whole blood samples, prepared as described

before in Section 2.3, were sonicated for 15 min at room

temperature and then submitted in parallel to SPE with

Chem Elut and Bond Elut Certify columns.

Calibration curves were prepared with five methanolic

standard solutions of these therapeutic drugs. The concen-

trations for mirtazapine, chlorpromazine, methotrimepra-

zine, clothiapine, olanzapine, clozapine, and thioridazine

were 0.5, 1, 5, 15 and 30 mg/L, while the concentrations

for haloperidol were 2, 4, 20, 60 and 120 mg/L, respec-

tively. The concentration of prazepam (internal standard)

was fixed at 5 mg/L. Drugs to prazepam area ratios were

measured, and the calibration curves were generated from

least-squares linear regression. The regression lines were

used to calculate the absolute recoveries (n = 6) of indi-

vidual drugs from spiked blood at three concentration

levels.

The intra-assay precision was assessed at three concen-

trations, 0.1, 0.5 and 2.0 mg/L for all the compounds, except

haloperidol, that was studied at 0.4, 2.0 and 8.0 mg/L. They

were extracted and analyzed on the same day (n = 6). The

inter-assay precision was assessed by analyzing a set of 10


spiked blood samples at the three concentrations on different

days, by different operators, using fresh methanolic calibra-

tion curves.

The limits of detection (LODs) and quantitation (LOQs)

were determined as the lowest concentration giving a

response of three times and 10 times, respectively, the

average of the base line noise defined from six control

samples.

The linearity of the method for mirtazapine, chlorpro-

mazine, methotrimeprazine, clothiapine, olanzapine, cloza-

pine, and thioridazine, was checked by preparing six

replicates of the blood calibration curves at three different

concentrations, ranging from 100 to 2000 mg/L (from 400 to

8000 mg/L for haloperidol), by addition of known amounts

of each drug to human whole blood.

3. Results and discussion

3.1. Chromatography

The Bond Elut Certify columns provided cleaner extracts

without interference of endogenous substances compared to

the Chem Elut columns. Figs. 2 and 3 show representative

GC-NPD chromatograms of extracted human whole blood

for each extraction method. Good chromatographic results

with reliable separations of these eight psychiatric drugs and

the chromatographic standard were achieved under the

chromatographic conditions described in relatively short

chromatographic times (16 min). Excellent chromato-

graphic behavior with sharp and symmetrical peaks without

derivatization was also obtained.

The selectivity of the method was studied for possible

matrix influence and for the possible interference of other

substances that can be present in combination with the

studied drugs. The influence of the matrix was studied by

analyzing a series of different pools of human whole blood

(n = 6) and checking for the presence of any interfering

substances at the retention time of the compounds of interest.

The absence of interferences from the matrix was further

confirmed by the daily use of the method with many different

samples. The potential interference of other drugs was

investigated by checking the retention time of the studied

drugs and other drugs, potentially detectable under the

described chromatographic conditions (Table 1). Mirtaza-

pine were found to co-elute with desipramine and elute close

to doxepin, causing peaks not to be completely resolved. In

real cases in which these compounds were present simulta-

neously, quantification can be accomplished using other

technique, such as GC–MS mode SIM or HPLC-DAD.

It is also interesting to report that both extraction pro-

cedures provided chromatograms without interferences in

the areas corresponding to the retention time of the studied

compounds and the internal standards. Caffeine, a common

beverage used in the population, did not interfere with the

studied drugs.

Mepivacaine was added as extraction internal standard to

control the extraction procedures. Mepivacaine, an uncom-

mon prescribed drug, presents good extraction recoveries

(100% with a relative standard deviation of 5%) using both

SPE procedures. Furthermore, this compound does not

interfere with a large variety of abused and therapeutic

drugs. Prazepam was chosen as chromatographic internal

standard because of its similar molecular structure and

physicochemical behavior. It is not widely used or pre-

scribed. The chromatographic internal standard was added

after the extraction, but prior to the introduction of the

extract into the gas chromatograph, in order to correct for

differences in the final volume of the concentrated extract

and injection volumes and to check chromatographic beha-

vior. The use of mepivacaine as extraction internal standard

(surrogate) and prazepam as chromatographic standard

allowed us to control the whole analytical procedure during

the routine performance of STA.

The nitrogen-containing structures of mirtazapine chlor-

promazine, methotrimeprazine, clothiapine, olanzapine, clo-

zapine, haloperidol, and thioridazine together with the use of

SPE columns are responsible for the high sensitivity with

minimal interference obtained using a nitrogen phosphorus

detector.

3.2. Comparison of the efficiencies of the two

SPE procedures

According to our experience in the routine practice in our

laboratory, both SPE methods under these chromatographic

conditions allow the determination of a wide variety of drugs

in the performance of STA. Evaluating their efficiencies is

one of the main reasons we have conducted this comparative

validation study.

A summary of the most representative analytical para-

meters studied are shown in Table 2. The accuracy of the

methods was expressed as percent recovery. Recoveries for

these therapeutic drugs using Chem Elut columns at 500 and

2000 mg/L (2000 and 8000 mg/L for haloperidol) were in the

range 21–65% with intra-assay and inter-assay precisions of

less than 17% and 19%, respectively. LODs and LOQs for

mirtazapine, chlorpromazine, methotrimeprazine, clothiapine,

olanzapine, clozapine, and thioridazine ranged from 62 to

161 mg/L and from 205 to 531 mg/L, respectively. LOD and

LOQ for haloperidol were 442 and 1458 mg/L, respectively.

Recoveries of these compounds using Bond Elut Certify

columns at 500 and 2000 mg/L (2000 and 8000 mg/L for

haloperidol) were in the range 44–97% with intra-assay

and inter-assay precisions of less than 7% and 14%, respec-

tively. LODs and LOQs for mirtazapine, chlorpromazine,

methotrimeprazine, clothiapine, olanzapine, clozapine, and

thioridazine ranged from 37 to 66 mg/L and from 122 to

218 mg/L, respectively. LOD and LOQ for haloperidol were

156 and 515 mg/L, respectively. Excellent linearity was

observed in the studied range for all tested therapeutic drugs,

r2 > 0.999 (n = 6).

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Fig. 2. Representative GC-NPD chromatograms of human whole blood after extraction using Chem Elut columns. (A) Blank whole blood, I = caffeine. (B) Blood spiked with 0.5 mg/L of each

therapeutic drug, I = caffeine, 1 = mepivacaine (extraction standard), 2 = mirtazapine 3 = chlorpromazine, 4 = methotrimeprazine, 5 = prazepam (chromatographic standard), 6 = clothiapine,

7 = olanzapine, 8 = clozapine, 9 = haloperidol, and 10 = thioridazine.

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Fig. 3. Representative GC-NPD chromatograms of human whole blood after extraction using Bond Elut Certify columns. (A) Blank whole blood, I = caffeine. (B) Blood spiked with 0.5 mg/L of

each therapeutic drug, I = caffeine, 1 = mepivacaine (extraction standard), 2 = mirtazapine, 3 = chlorpromazine, 4 = methotrimeprazine, 5 = prazepam (chromatographic standard), 6 = clothia-

pine, 7 = olanzapine, 8 = clozapine, 9 = haloperidol, and 10 = thioridazine.


Table 1

Retention times (absolute and relative to diazepam) of drugs detect-

able under the described chromatographic conditions

Compound RTabsolute (min) RRT

Fenfluramine 1.47 0.05

Chlomethiazole 1.48 0.05

Ephedrine 1.66 0.08

Piracetam 1.82 0.10

Amphepramone 1.95 0.12

Pyrithyldione 1.95 0.12

Fenproporex 2.18 0.16

Metronidazole 2.23 0.17

Acetaminophen 2.37 0.19

Methylphenidate 2.78 0.25

Meperidine 2.89 0.27

Caffeine 3.13 0.30

Theobromine 3.21 0.31

Phenazone 3.39 0.34

Viloxazine 3.45 0.35

Thiopental 3.46 0.35

Fluoxetine 3.50 0.36

Diphenhydramine 3.54 0.37

Lidocaine 3.60 0.37

Fluvoxamine 3.62 0.38

Oxprenolol 3.77 0.40

Aminopyrine 3.78 0.40

Doxylamine 3.85 0.41

Theophylline 3.88 0.42

Propyphenazone 3.99 0.43

Phenobarbital 4.01 0.44

Tramadol 4.07 0.45

Trimetazidine 4.32 0.48

Chlorpheniramine 4.48 0.51

Cyclizine 4.64 0.53

Metoprolol 4.64 0.53

Clenbuterol 4.80 0.56

Mepivacaine 4.92 0.58

Fluconazole 5.02 0.59

Venlafaxine 5.04 0.59

Ticlopidine 5.15 0.61

Brompheniramine 5.20 0.62

Dextromethorphan 5.48 0.66

Propranolol 5.53 0.67

Methadone 5.57 0.68

Primidone 5.78 0.71

Atropine 5.85 0.72

Etamiphyllin 5.88 0.72

Propoxyphene 5.92 0.73

Mianserin 5.93 0.73

Amitriptyline 5.94 0.73

Nortriptyline 6.04 0.75

Levorphanol 6.05 0.75

Pirlindole 6.07 0.75

Moclobemide 6.10 0.76

Imipramine 6.14 0.76

Trimipramine 6.14 0.76

Medazepam 6.17 0.77

Pyrilamine 6.18 0.77

Doxepin 6.18 0.77

Desipramine 6.25 0.78

Table 1 (Continued )


Mirtazapine 6.25 0.78

Cyclobenzaprine 6.26 0.78

Trihexyphenidyl 6.37 0.80

Halazepam 6.46 0.81

Pentazocine 6.56 0.83

Biperiden 6.59 0.83

Timolol 6.60 0.83

Bupivacaine 6.63 0.84

Melitracen 6.63 0.84

Phenytoin 6.75 0.86

Alimemazine 6.76 0.86

Oxazepam 6.85 0.87

Bisoprolol 7.01 0.90

Maprotiline 7.01 0.90

Cyproheptadine 7.18 0.92

Phenylbutazone 7.20 0.92

Sertraline 7.23 0.93

Codeine 7.25 0.93

Bromhexine 7.33 0.94

Buflomedil 7.33 0.94

Dothiepine 7.37 0.95

Pentoxyphilline 7.40 0.96

Lorazepam 7.47 0.97

Citalopram 7.51 0.97

Lamotrigine 7.57 0.98

Tetrazepam 7.62 0.99

Cloperastine 7.63 0.99

Clomipramine 7.64 0.99

Diazepam 7.69 1.00

Nordiazepam 8.10 1.06

Chlordiazepoxide 8.21 1.08

Chlorpromazine 8.25 1.09

Flumazenil 8.25 1.09

Methotrimeprazine 8.52 1.13

Clobazam 8.55 1.13

Trimethoprim 8.64 1.14

Paroxetine 8.80 1.17

Midazolam 8.90 1.18

Bromazepam 8.93 1.19

Loxapine 8.95 1.19

Flunitrazepam 8.97 1.20

Metoclopramida 9.12 1.22

Ketotifen 9.15 1.22

Chloroquine 9.18 1.23

Amoxapine 9.21 1.23

Ambroxol 9.25 1.24

Prazepam 9.34 1.25

Laudanosine 9.52 1.28

Trifluoperazine 9.66 1.30

Clothiapine 9.71 1.31

Olanzapine 9.90 1.34

Zolpidem 10.36 1.41

Flurazepam 10.37 1.41

Quinidine 10.43 1.42

Quinine 10.44 1.42

Tiapride 10.47 1.42

Clozapine 10.94 1.50

Hydroxyzine 11.05 1.51

Alprazolam 11.41 1.57


Table 1 (Continued )


Haloperidol 11.48 1.58

Diltiazem 11.53 1.59

Cinnarizine 12.45 1.73

Etoperidone 12.57 1.74

Sulpiride 12.75 1.77

Thioridazine 12.80 1.78

Verapamil 13.13 1.83

Trifluoperazine 13.68 1.91

Periciazine 13.74 1.92

Trazodone 14.06 1.97

Lofepramine 14.09 1.98

Sulforidazine 14.85 2.09

Clebopride 15.46 2.19

Based on the obtained data, the use of the Bond Elut

Certify columns resulted in more advantages besides the

detection of therapeutic drugs, such as better and more easily

reproducible recoveries, cleaner extracts, better sensitivity,

and less solvent consumption and disposal.

Table 2

Extraction recovery (%), intra-assay and inter-assay precision (R.S.D. %), li

eight psychotropic drugs in whole blood using Chem Elut and Bond Elu

Compound Recovery (%)

mean (n = 6)

Intra-assay

precision

R.S.D. (%) (n = 6)

In

pr

R.

0.5 mg/L 2.0 mg/L 0.5 mg/L 2.0 mg/L 0.

Mirtazapine

Chem Elut 65 64 10 9 12

Bond Elut Certify 90 92 2 5 4

Chlorpromazine

Chem Elut 21 22 12 10 15


Methotrimeprazine

Chem Elut 21 23 12 6 15


Clothiapine

Chem Elut 33 34 17 11 19


Olanzapine

Chem Elut 42 38 11 9 15


Clozapine

Chem Elut 46 47 14 9 18


Haloperidola

Chem Elut 36 29 11 10 13


Thioridazine

Chem Elut 21 23 13 9 19


a Two milligram per liter for haloperidol.

3.3. Considerations regarding strategy in the

performance of STA

It is crucial to have validated analytical methods in order

to get accreditation in a forensic or clinical laboratory.

Toxicologists must have absolute confidence in their proce-

dures in order to uphold toxicological results in courts of

law. Furthermore, STA always requires well-characterized

and validated methods in order to produce reliable results

that can be interpreted satisfactorily. Moreover, STA drug

concentrations are low and biological matrix are usually not

clean.

The Chem Elut extraction procedure, with at least two

columns: one for the acidic and neutral substances and other

for basic and neutral substances, has been routinely used in

our laboratory for sample pre-treatment and clean-up of

biological samples in the last recent years. The use of the

Bond Elut extraction procedure, using just one column, as

regular part of drug testing has been introduced in our

laboratory, after developing several series of comparative

nearity (r2), limits of detection (LOD) and quantitation (LOQ) for the

t Certify columns and analyzed by GC-NPD

ter-assay

ecision

S.D. (%) (n = 10)

Linearity r2

(n = 6)

Limit of

detection

LOD (mg/L)

(n = 6)

Limit of

quantitation

LOQ

(mg/L) (n = 6)5 mg/L 2.0 mg/L

11 0.999 63 208

7 0.999 45 148

11 0.999 139 459

10 0.999 46 152

11 0.999 122 403

10 0.999 52 172

13 0.999 65 214

5 0.999 46 152

11 0.999 123 406

10 0.999 66 218

10 0.999 62 205

5 0.999 37 122

15 0.999 442 1458

5 0.999 156 515

12 0.999 161 531

10 0.999 62 205


Table 3

List of therapeutic, toxic, and fatal levels for the studied psychiatric drugs published in the literature

Compound Therapeutic level (mg/L) Toxic level (mg/L) Fatal level (mg/L) Reference

Mirtazapine (serum) 0.02–05 N.A.a N.A. [27]

Chlorpromazine (blood) 0.1–2 0.8–3 >1 (mean = 5) [28]

Methotrimeprazine (serum) 0.03–0.15 0.5 N.A. [27]

Clothiapine N.A. N.A. N.A. –

Olanzapine (serum) 0.01–0.05 (0.1) 0.2 N.A. [27]

Clozapine (serum) 0.1–0.6 0.8–1.3 3 [27]

Haloperidol (blood) 0.0005–0.0009 0.05–0.5 N.A. [28]

Thioridazine (blood) 0.05–5 1–5 >2 (mean = 5) [28]

a Not available.

Fig. 4. GC-NPD chromatogram of whole blood obtained from an

attempted suicide after extraction using a Bond Elut Certify column.

Detected are, 1 = caffeine, 2 = mepivacaine (extraction standard),

3 = methotrimeprazine, 4 = midazolam, 5 = prazepam (chromato-

graphic standard), 6 = thioridazine, 7 = mesoridazine, and 8 = sul-

foridazine.

studies with different types of drugs regarding these two SPE

procedures [20,22,23].

The combination of the chromatographic techniques is

highly recommended to achieve the most comprehensive

drug screen. Knowing that some drugs cannot be detected in

the GC-NPD screening, or show poor chromatographic

properties or display thermal instability during injection,

it is more convenient to have a single sample extract to

complete the screening or confirmation. This was the reason

we used the Bond Elut Certify columns since both acidic-

neutral and basic eluates were obtained.

3.4. Utility of the method

The utility of the methods is demonstrated comparing the

literature levels with the LODs obtained in this study.

According to therapeutic and toxic situations published in

the literature [27,28] and shown in Table 3, our assay using

the described Bond Elut Certify method, of choice, allows

detection and quantitation of mirtazapine, chlorpromazine,

methotrimeprazine, clothiapine, clozapine, and thioridazine

at therapeutic levels. Olanzapine can only be detected at high

therapeutic and toxic concentrations. It is important to

considerate that the olanzapine data published in the litera-

ture refers to serum instead whole blood. Haloperidol can

only be detected at toxic concentrations. Not all of the drugs

levels are available in Table 3 since some of the studied

drugs have been recently introduced in the market with

subsequent lack of data.

3.5. Application to a case history

The SPE method of choice, the Bond Elut Certify

procedure, was applied to the analysis of the blood sample

collected in ICU from a 20-year-old woman who tried to

commit suicide by ingestion of Meleril1 (thioridazine). The

woman, who suffered schizophrenia and had a previous

history of drug abuse, manifested a Glasgow coma score

of 7 on admission to hospital. According to our compre-

hensive toxicological screening protocol the blood sample,

was previously tested for abuse drugs on a Hitachi 902

Automatic Analyzer using Cedia1 reagents, and for ethanol

and other volatiles on a headspace GC-FID equipment. The

results were negative for all abuse drugs including ethanol.

Fig. 4 shows the chromatogram of the Bond Elut Certify

blood extract, demonstrating the good performance of the

method. After screening with the method described here,

methotrimeprazine, thioridazine, mesoridazine, sulforida-

zine (both psychoactive thioridazine metabolites), and mid-

azolam, a sedative agent administrated in the hospital, were

detected and quantified using GC-NPD and confirmed using

GC–MS mode TIC (total ion chromatogram), under the

same chromatographic conditions. The toxicological find-

ings are shown below in Table 4.


Table 4

Toxicological findings in case history

Bond Elut Certify procedure Blood concentrations

(mg/L)

Methotrimeprazine <LOQ

Thioridazine 1.6

Mesoridazine 0.4

Sulforidazine 0.3

4. Conclusions

In conclusion, the use of Bond Elut Certify mixed-phase

columns resulted in numerous advantages for the detection

of eight psychiatric drugs including improved and more

reproducible recoveries, cleaner extracts, better sensitivity

and reduced solvent consumption, and represents an excel-

lent alternative to traditional Chem Elut columns.

Consequently, it is concluded that the combination of

Bond Elut Certify columns for extraction of blood samples

and GC-NPD for detection provides a sensitive and repro-

ducible method of choice which allows the determination of

therapeutic and toxic concentrations of mirtazepine, chlor-

promazine, methotrimeprazine, clothiapine, olanzapine, clo-

zapine, and thioridazine, and toxic concentrations of

haloperidol in blood. These results are in accordance with

our previous studies and complete the analytical data for the

detection of psychotropic drugs [20,22,23].

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